Relevance to public health: Alcoholism is an international public health concern that arises from a combination of genetic and environmental factors. Identification of the gene(s) that contribute to vulnerability for developing alcohol dependence can greatly improve efforts to treat and prevent this damaging disease. The broad objective of this application is to assess whether oxidative stress is a mechanism by which a defined genomic region in mice directly influences alcohol physical dependence and associated alcohol withdrawal, complex traits associated with alcoholism in humans. We previously fine mapped to 1.1 Mb on distal mouse Chr 1 a Quantitative Trait Locus (QTL) that underlies 13% of individual variation in ethanol withdrawal severity using inbred and congenic strains. Analyses of genotype-dependent variation in protein product sequences and mRNA expression for revealed that three genes in this region code for proteins known to function in oxidative stress pathways, two of which are protein subunits of complex I and II of the mitochondrial respiratory chain. In Aims 1 and 2, we will compare brain oxidative stress levels and respiratory complex activity of congenic and background mice during withdrawal from both acute and chronic ethanol. Protein carbonyl content will be measured in whole brain samples using a colorimetric DNP assay adapted for ELISA as a reliable and quantitative biomarker of oxidative stress. Respiratory chain complex amount and activity will be assessed using a combination of blue native PAGE (BN-PAGE) and histochemical techniques. Whole protein complexes will be resolved from mitochondria-enriched whole brain fractions on gels that allow their retention of native conformation and activity levels when exposed to appropriate substrates. Densitometry is used for relative quantitation. We will compare these biochemical measures between naive, ethanol-dependent, and ethanol-withdrawing mice of different genetic makeup to assess the involvement of a mitochondrial/oxidative stress mechanism in the behavioral expression of severe alcohol withdrawal. In Aim 3, we will extend these principals to directly test in vivo the hypothesis that an antioxidant (melatonin) is effective in attenuating alcohol withdrawal through its protective effects on oxidative stress levels and mitochondrial respiratory activity.